Starostik, P. | bioRxiv Science

bioRxiv2026

ONETest PathoGenome: A Multi-Cohort Evaluation of an Optimized NGS Assay for Detection of Lower Respiratory Pathogens in Bronchoalveolar Lavage

BackgroundLower respiratory tract infections (LRTIs) remain diagnostically challenging when culture and molecular assays are negative or delayed. We evaluated ONETest Pathogenome (OT), an automated hybrid-capture metagenomic assay with core-genome enrichment probes, for direct pathogen detection in bronchoalveolar lavage (BAL). MethodsAnalytical performance (LoD, precision, continuity) was assessed using whole-cell spike-ins into culture-negative BAL fluid. Technical performance was assessed in 119 specimens profiled by OT and whole-metagenome shotgun sequencing (WmGS, cohort 1). Clinical accuracy was evaluated in 360 specimens (cohort 2) benchmarked against routine bacterial and acid-fast bacillus (AFB) culture. Laboratory-developed test (LDT) validation included 43 specimens (cohort 3) benchmarked to bacterial and AFB culture. ResultsOT uses 6.2 million probes covering core genomes across 50 microbial families (>250 respiratory pathogens). In BAL specimens, OT increased normalized on-target microbial abundance 26-fold versus that of WmGS while preserving within-sample microbial diversity. In cohort 2, OT achieved species-level sensitivity of 80% and specificity of 99% across culture-confirmed isolates and detected [≥]1 culture-confirmed organism in 100/115 culture-positive specimens (87%), while applying species-specific background baselines to mitigate overcalling. Additive yield was 21% (76/360), with 7.5% (27/360) of specimens having [≥]1 additional finding supported by orthogonal testing. In LDT validation, OT identified [≥]1 culture-confirmed organism in 34/40 culture-positive specimens (85%) with one OT-positive/culture-negative specimen. ConclusionsOT is an assay with a turnaround time <24 h complementary to culture that improves pathogen detection and expands microbiologic findings through additional detections and co-detections, including slow-growing organisms that may require prolonged incubation by conventional methods.

microbiology↗

bioRxiv2018

Co-occurring genetic alterations in the RAS pathway promote resistance to MET inhibitor treatment in non-small cell lung cancer with a MET exon 14 skipping mutation

PURPOSEWhile patients with advanced-stage non-small cell lung cancers (NSCLCs) harboring MET exon 14 skipping mutations (METex14) often benefit from MET tyrosine kinase inhibitor (TKI) treatment, clinical benefit is limited by primary and acquired drug resistance. The molecular basis for this resistance remains incompletely understood.\n\nMETHODSTargeted sequencing analysis was performed on cell-free circulating tumor DNA obtained from 289 patients with advanced-stage METex14-mutated NSCLC.\n\nRESULTSProminent co-occurring RAS-MAPK pathway gene alterations (e.g. in KRAS, NF1) were detected in NSCLCs with METex14 skipping alterations as compared to EGFR-mutated NSCLCs. There was an association between decreased MET TKI treatment response and RAS-MAPK pathway co-occurring alterations. In a preclinical model expressing a canonical METex14 mutation, KRAS overexpression or NF1 downregulation hyperactivated MAPK signaling to promote MET TKI resistance. This resistance was overcome by co-treatment with crizotinib and the MEK inhibitor trametinib.\n\nCONCLUSIONOur study provides a genomic landscape of co-occurring alterations in advanced-stage METex14-mutated NSCLC and suggests a potential combination therapy strategy targeting MAPK pathway signaling to enhance clinical outcomes.

cancer biology↗